Gene Search Vector and Gene Search Method

ABSTRACT

The present invention provides a gene search method comprising P-element insertion and being capable of efficiently identifying a novel gene regulating various biological functions of Drosophila and specifying a function corresponding to the novel gene. An unknown gene of Drosophila can be searched by the method comprising a step of inserting in the genome of Drosophila a gene search vector carrying two sets of an expression regulatory sequence comprising UAS sequence for GAL4 transcription factor GAL4 and a promoter sequence, as integrated in the P-element sequence in such a manner that their downstreams are in opposite directions, mating the vector-inserted Drosophila with a Drosophila expressing the GAL4 to create progeny individuals, and identifying a vector-inserted line with a phenotype different from those of wild-type Drosophila, and determining the nucleotide sequence of the gene for the mutant phenotype.

FIELD OF THE INVENTION

[0001] The present invention relates to a novel gene search vector forefficiently detecting an unknown gene of a fly, Drosophila, and a genesearch method using the vector.

DESCRIPTION OF THE RELATED ART

[0002] Rapid progress has been made in the determination of thefull-length genome sequences of various species. So as to effectivelyutilize the information about these genome sequences, a currentinvestigative issue is the identification of a specific sequence domainpractically responsible for a biological function to sequentiallydetermine such domains in the form of “gene” corresponding to biologicalfunction.

[0003] The essential mechanisms of biological functions are preserved inany species, and genes allowing the mechanisms to function are alsoanalogous in any species. Based on such grounds, the functionalelucidation of a gene of a model lower organism is applicable to othernumerous organisms including humans. The gene is for example life-spanregulatory gene. The evidence up to date indicates that life spancharacteristically has a close relation with stress resistance,anti-oxidative action and biological protective potency. Because thesefunctions are regarded to belong to the principal mechanisms of anyspecies, the elucidation of the mechanisms in a model organism and thefindings about the factors and genes involved in the mechanisms are ofsignificant importance in various industries. For example, it isexpected that life-span prolonging factor and stress-resistance factornot only provide information useful for the development of medicinalproducts but also are directly involved in the development of foodstuffshelping and supporting the control and promotion of human health(foodstuffs enriched with such factors and foodstuffs containingingredients facilitating the generation of these factors in livingorganisms). It is also expected that industrially useful animals andplants, given with these ingredients in the forms of feeds orfertilizers or integrated with genes encoding these ingredients, canyield an improved productivity. Furthermore, factors capable ofregulating the appearance and growth of insects or the propagatingactions thereof provide new strategies for the control of thereproduction of useful insects or harmful insects with deep relation toagriculture and forestry. Otherwise, factors triggering cell growth ordifferentiation and abnormal actions provide valuable information forthe molecular understanding of the fundamental pathogenesis of humangenetic diseases or the functions of organisms; and these factorsadditionally serve as potent materials for the therapies of cancer andnerve diseases and for the development of novel drugs for thesediseases.

[0004] Drosophila has traditionally been used frequently as a materialfor genetic research works and is endowed with powerful analyticalmodalities in the fields of genetics and molecular biology. Thus,Drosophila has been acclaimed as an excellent model organism forsearching novel gene. In a practical sense, many novel genes including“homeobox gene” have been isolated from Drosophila.

[0005] The mutagenesis process comprising inserting the transposonP-element into the genome has been known as the means for searchingnovel genes in Drosophila (Science 239: 1121-1128, 1988; Proc. Natl.Acad. Sci. USA 92:10824-10830, 1995).

[0006] However, the frequency of the occurrence of a homozygotephenotype recovered through such mutagenesis using P-element is as lowas about 15%, which is not sufficient for the detection of a gene in thegenome (Science 239:1121-1128, 1988). It is considered that this is dueto the facts that the sites for P-element insertion are mostly in theupstream of the coding sequence (Proc. Natl. Acad. Sci. USA92:10824-10830, 1995) and that the genome per se is functionallyredundant (Cell 86:521-529, 1996). Additionally, it is also indicatedthat the resulting phenotype sometimes has no relation with P-elementinsertion (Genetics 147:1697-1722, 1997).

[0007] As means for the expression of a gene of Drosophila in anenforced fashion, further, a method has been known, comprising insertingin the Drosophila genome a vector carrying UAS (upstream activatorsequence) enhancer as the target sequence of a yeast-derived GAL4transcription activator, mating the transformed Drosophila with aDrosophila capable of expressing GAL4 (GAL4 expression line) to create aprogeny individual, and allowing the progeny individual to express agene downstream the UAS enhancer (Development 118:401-415, 1993).

[0008] Another method using a combination of the GAL4-UAS enhancercompulsory expression system and the mutation induction with P-elementinsertion has been known (Proc. Natl. Acad. Sci. USA 93:12418-12422,1996; Dros. Inf. Serv. 80:90-92, 1997). According to the method, a pairof the UAS enhancer/promoter is integrated in the multicloning site of aP-element transformation vector pCa SpeR (Gene 74:445-456, 1988) on theside of 3′P-element; Drosophila with the vector inserted therein ismated with the GAL4 expression line, to enforce the expression of genesdownstream the vector insertion site in a progeny individual to identifyphenotypes and the genes corresponding thereto.

[0009] The method using a combination of the GAL4-UAS enhancer forcedexpression system and the mutagenesis with P-element insertion isexcellent in that genes can be detected on the basis of gain-of-functionphenotype and loss-of-function phenotype. However, the occurrence of themutant phenotype caused by P-element insertion is so low by the method.Hence, means or methods capable of efficiently allowing a mutantphenotype to emerge have been desired so as to detect a great number ofuseful novel genes.

[0010] In such circumstances, the invention has been accomplished forthe purpose of providing a novel gene search vector capable ofefficiently identifying a novel Drosophila gene in a manner so as tospecify the function corresponding to the novel Drosophila gene; and agene search method.

SUMMARY OF THE INVENTION

[0011] The present invention provides a gene search vector, whichcarries P-element sequences and two sets of an expression regulatorysequence comprising a UAS sequence for GAL4 transcription activator anda promoter sequence, wherein the expression regulatory sequences areintegrated in the P-element sequence in such a manner that theirdownstreams are in opposite directions.

[0012] In one preferable embodiment of the gene search vector, thepromoter is the core promoter of heat shock protein hsp70 gene.

[0013] In the other preferable embodiment of the gene search vector, thetwo sets of the expression regulatory sequence are independentlyintegrated in the 5′P-element sequence and 3′P-element sequence.

[0014] In another preferable embodiment of the gene search vector, amarker gene is present between the two sets of the expression regulatorysequence, while Drosophila white gene is used as the marker gene.

[0015] In a still further embodiment of the gene search vector, the fulllength of the vector DNA is less than 8 kbp.

[0016] The present invention also provides a fly carrying the genesearch vector set forth above being integrated in the genome thereof.

[0017] The present invention further provides a method for searching anunknown gene of Drosophila, comprising the steps of:

[0018] (a) inserting the gene search vector of any one of claims 1 to 6into the genome of Drosophila,

[0019] (b) mating the vector-inserted Drosophila with a Drosophilaexpressing the GAL4 transcription activator to create progenies, andidentifying a vector-insertion line with a phenotype different fromthose of wild-type Drosophila, and

[0020] (c) determining the nucleotide sequence of the gene for thephenotype of the mutant individual.

[0021] In one preferable embodiment of the gene search method, in thestep (c), a mRNA fragment including mRNA transcribed from a partialsequence of the gene search vector is amplified byreverse-transcriptase-polymerase chain reaction method to determine thenucleotide sequence of the resulting amplified DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a schematic view depicting the inventive method; andFIG. 2 shows the comparison in the putative amino acid sequences of Rap2gene and mGST gene between humans and Drosophila.

DETAILED DESCRIPTION OF THE INVENTION

[0023] As shown in FIG. 1a, for example, the gene search vector(sometimes referred to as “GS vector” hereinafter) comprises two sets ofan expression regulatory sequence of the UAS enhancer and an appropriatepromoter sequence (for example, the core promoter of heat shock proteinhps70 gene), as integrated in the P-element sequence in such a mannerthat the downstreams are in opposite directions. Compared withconventional such vectors (Proc. Natl. Acad. Sci USA 93:12418-12422,1996; Dros. Inf. Serv. 80:90-92, 1997), the inventive method using suchtwo sets of the expression regulatory sequence can permit the emergenceof a mutant phenotype at a two-fold frequency. Such GS vector can beprepared from for example P-element transformation vector pCaSpeR3 (Gene74:445-456, 1988). Using pCaSpeR3, two sets of the expression regulatorysequence can be inserted individually in the multicloning site on the3′P-element side, but preferably, one set of the expression regulatorysequence is inserted in a P transposase-encoding region appropriatelyprepared on the side of 5′P-element. A marker gene is integrated betweenthe two sets of the expression regulatory sequence. The marker gene, acausative gene of the change in the Drosophila phenotypes, canaccomplish ready screening of an individual with the GS vectorintegrated in the genome thereof, on the basis of the change in thephenotypes. As the marker gene, appropriate ones such as white gene inFIG. 1a can be used.

[0024] The GS vector is recommended to constructed at a full lengthbelow 8 kbp, more preferably at a full length of 5 to 6 kbp. The vectorof a smaller size enables the improvement of the insertion efficiency,to raise the frequency of the occurrence of a mutant phenotype.

[0025] By the method of such composition described above, the GS vectorprepared below in Example 1 can allow the emergence of a phenotype at afrequency about 10-fold the frequency caused by the vector prepared bythe conventional method (Proc. Natl. Acad. Sci. USA 93:12418-12422,1996).

[0026] According to the method of the present invention, the GS vectorthus prepared is inserted in the genome of Drosophila to crease pluralGS vector insertion lines. The GS vector is randomly inserted in varioussites of the genome, owing to the presence of the P-element. As shown inFIG. 1b, then, the GS vector insertion lines and the GAL4 expressionlines are mated. Various lines are known as the GAL4 expression lines,so one of such lines is appropriately selected.

[0027] In the progeny individuals from the mating, the sequencesdownstream the GS vector insertion sites are compulsively transcribedand expressed in the GAL4 expressing tissues. Then, the full-length mRNAis transcribed when the GS vector insertion site is located on 5′ sideof the gene; and antisense RNA and mRNA encoding an incomplete-lengthprotein are transcribed when the GS vector insertion site is locatedinside the gene; antisense RNA is transcribed when the GS vectorinsertion site is located on the 3′ side of the gene, Further, thebiological actions of these transcription products are exerted asphenotypic changes in the progeny individuals. Sense-chain transcriptionis exerted as a phenotype with functional acquisition due to the ectopicexpression of the resulting translation product; and antisense RNAtranscription is exerted as a functionally defective phenotype due tothe inhibition of the translation of a wild-type product. Additionally,a partially defective protein generated indicates the occurrence of amutant of a type, dominant active or dominant negative. The site andtiming with the occurrence of forced expression depend on the expressionspecificity of GAL4. By selecting a GAL4 expression line for mating,forced expression can be induced, in a manner specific to the specifictissue or timing.

[0028] Because the function of a gene significantly varies, depending onthe cell biological background for the expression of the gene,additionally, a conventionally unknown potential function of the genecan sometimes be exhibited, for the first time, when the gene iscompulsively expressed at a site different from the essential expressionsite.

[0029] According to the method of the present invention, a novel genecan be detected by isolating a causative gene on the basis of a mutantphenotype recovered by compulsive expression. As shown in FIG 1 c, forexample, the gene can be detected by amplifying the compulsivelyexpressed mRNA by reverse-transcriptase-polymerase chain reaction method(RT-PCR) and isolating the DNA sequence from the resulting PCR product.As the RT-PCR template, a mRNA fragment comprising a partial sequence ofthe GS vector is satisfactorily isolated by using polyA(+) RNA.

[0030] According to the method of the present invention, the GSvector-inserted individuals are used as they are for the bioassay forthe screening of all the biological functions such as development,growth, behavior and life span, to cover the detection of novel genesinvolved in the biological functions. Using the Drosophila gene thusdetected or a partial sequence thereof as probe, a homologous gene canbe isolated from the genome library of an organism other thanDrosophila, the cDNA library thereof or the whole genome sequenceinformation. Otherwise, a homologous gene can be amplified and isolatedby PCR using the genome DNA or cDNA of an organism other thanDrosophila.

[0031] The invention will now be described in more detail in moreconcrete manner in the following Examples, but the invention is notlimited to these Examples.

EXAMPLE 1

[0032] According to the gene search method of the present invention, theGS vector was inserted in Drosophila, to prepare GS vector insertionlines.

[0033] 1. Construction of GS vector

[0034] By inducing a mutation in P-element transformation vectorpCaSpeR3 (Gene 74:445-456, 1988) by means of PCR, EcoRI site wasprepared in the P transposase-encoding region in the vicinity of the 5′Pterminus. Five tandem repeats of UAS conjugated with a core promoterderived from the gene hps70 of pUAST (Development 118: 401-415, 1993)were inserted at the EcoRI site in the vicinity of the 5′ P terminus andthe XhoI site at the multicloning site in the vicinity of the 3′ Pterminus, to prepare GS vector of a schematic structure shown in FIG.1a. The GS vector of its 5245-bp full length carries the white gene as amarker gene between 2 sets of UAS enhancer/promoter.

[0035] 2. Preparation of GS vector insertion lines

[0036] The GS vector prepared above in 1 was introduced in Drosophila(yw line; supplied from the Tokyo Metropolitan University), to preparethe transgenic Drosophila. By using a transposase delta2-3,subsequently, the GS vector was transposed, to prepare GS vectorinsertion lines of 613 in number. The transposed GS vector was locatedin the chromosome X in 147 lines, the chromosome 2 in 226 lines, thechromosome 3 in 237 lines and the chromosome 4 in 3 lines. Additionally,at least one detectable phenotype (for example, lethality and defectiveadult structure) could be detected in 394 lines (64%).

EXAMPLE 2

[0037] Among the GS vector insertion lines prepared in Example 1, 163lines randomly selected from the 394 lines with any detectable phenotypewere mated with the GAL4 expression lines to generate progeny (F1)individuals, which were then allowed to express transcription productsby the GAL4 transcription activator. The resulting transcriptionproducts were analyzed. Herein, dpp-GAL4, sev-GAL4, hs GAL4, 29BD-GAL4and c355-GAL4 (all supplied by the Bloomington Stock Center) were usedas the GAL4 expression lines.

[0038] 1. Identification method of transcription products

[0039] Fly larvae of age 3 days from the F1 individual between the GSvector insertion lines and the hs GAL4 line as prepared in Example 1.2were placed at a ratio of 20-30 fly larvae per tube in 1.5-ml tubes,which were then warmed at 37° C. for one hour. From the larvae wasisolated polyA(+) RNA by using Quick prep micro mRNA purification kit(manufactured by Amersham Pharmacia Biotech Co. Ltd.), which wassubjected to mRNA reverse-transcription by using First-strand cDNAsynthetic kit (manufactured by Amersham Pharmacia Biotech Co. Ltd.).Using 1 μl of the resulting mRNA as template, 5′ P- and 3′ Ptranscription products were amplified by PCR with an enzyme mixtureELONGASE (manufactured by GIBCO BRL INC.). The total volume of the PCRsolution was 50 μl. Using as PCR primers the upstream common primer (SQID No. 1) and NotI-terminated oligo-d(T) primer (SQ ID No. 2), cDNA wassynthetically prepared. Subsequently, 1 μl of the first PCR product wasadded to the PCR solution in total of 50 μl, to separately amplify the5′ P transcription product and the 3′ P transcription product. As thePCR primers, an upstream primer 5′ P-specific primer (SQ ID No. 3) or 3′P-specific primer (SQ ID No. 4) and a downstream primer (SQ ID No. 5)were used. By using an auto-analyzer Perkin Elmer Gene Amp PCR System2400 or 9700, PCR was carried out under the following cyclingconditions: 94° C. (60 seconds); 16 cycles of 94° C. (15 seconds) and65° C. (10 minutes); 12 cycles of 94° C. (15 seconds) and 65° C. (10minutes; the duration is prolonged by 15 seconds per one cycle); and 72°C. (10 minutes). After termination of all the cycles, the resultingsolution was retained at 4° C. The resulting PCR products wereelectrophoresed on 1.0% agarose (Sigma type II) gel; bands of theamplified products were cut out with a knife; and the PCR products werepurified by QIAEX II gel extraction kit (manufactured by QIAGEN Co.).Along with ABI PRISM Cycle Sequencing Kit (manufactured by Perkin ElmerCo.), the purified DNA fragments were subjected to sequencing by using a5′-P/3′-P common primer (SQ ID No. 6) by an auto-analyzer ABI PRISMGENETIC Analyzer 310 (manufactured by Perkin Elmer Co.).

[0040] The sequence analogy was screened on the basis of the NCBInon-redundant nucleic acid data base and the dbest or NCBI non-redundantprotein data base by using the program BLASTN or BLASTX (Proc. Natl.Acad. Sci. USA 87:5509-5513, 1990).

[0041] 2. Analysis of transcription products

[0042] Because the P-element induces the occurrence of the repetition ofthe 8-bp target sequence at the inserted site (Cell 34:25-35, 1983), twodifferent transcription products derived from the single insertion ofthe P-element can readily be identified due to the presence of thecommon first (5′-terminal) 8 base pairs. Because 8 GS vector insertionlines expressed two transcription products with different terminalsequences, it was verified that two P-elements were inserted in the sameone chromosome. Owing to the P-element insertion including such doubleinsertion, transcription products in total of 171 were identified in thesame manner as in 1 and were then used for screening with data base.

[0043] The results shown in Table 1 indicate that 47% of the sequencedtranscription products corresponded to known sequences. TABLE 1 Numberof insertions % Known Sequences* known genes 35 20 ESTs 33 19 STSa 6 4Transposons or repetitive sequences 7 4 Novel sequences Homolog 2 1Novel 88 51 Total 171 100

[0044] The site of P-element insertion was examined in the transcriptioninitiation sites of known genes and the known ESTs (Expressed SequenceTags). The P-element insertion site was determined, relative to a knownmRNA 5′ terminus designated +1. The results are shown in Table 2 (knowngenes) and Table 3 (ESTs). 50% of the sites of P-element insertion werelocated at positions −150 to +100. Specifically, the sites were locatedmost frequently at positions −100 to −1. In terms of the relation withknown genes, P-element was inserted upstream the protein-encodingregions of 83% of the known genes, suggesting that many of thephenotypes detected by the screening were due to the excessiveexpression or ectopic expression of the full-length mRNA. TABLE 2Inserted locus Known genes GS line Full name Symbol Accession ScoreInsertion Site* 97 Transcription-factor-ll-S TfllA-S X83271 <1e-100 −6771029 Netrin-B NetB U60317 le-24 −95 1032 polyhomeotic ph M64750 <1e-100−353 1038 raspberry ras L14847 <1e-100 −666 1053 amnesiac amn U22825<1e-100 +2597 1069 Fasciclin 2 Fas2 M77165 4e-69 −63 1073 wingsapart-like wapl U40214 2e-84 −117 1091 embryonic lethal, abnormalversion elav M21153 <1e-100 −450 1115 bang senseless bss X89811 <1e-100+364 1131 Actin 5C Act5C X15730 <1e-100 −923 1141 armadillo arm X54468<1e-100 intron 1144 ovo ovo X59772 <1e-100 −58 2011 High mobilityprotein D HmgD M77023 <1e-100 −66 2042 High mobility protein D HmgDM77023 <1e-100 −10 2115 exuperantia exu S72757 <1e-100 3′ flankingregion 2120 fuzzy fy AF022891 <1e-100 −196 2137 High mobility protein DHmgD M77023 2e-11 intron 2141 string of pearls sop U01335 9e-21 −35 2160Glutathione S trasferase 2 Gst2 M95198 <1e-100 +10 2163B ornithinedecarboxylase antizyme AF038597 <1e-100 intron 2220 exuperantia exuS72757 <1e-100 3′ flanking region 2227 anterior open aop M97694 <1e-100−97 2228 expanded ex L14768 <1e-100 +617 3026 tramtrack ttk Z11723 2e-35−3 3029 Dihydroorotate dehydrogenase Dhod L00964 3e-37 −277 3052 HistoneH2A variant His2AvD X07485 <1e-100 intron 3069 tramtrack ttk Z117231e-43 intron 3089 neuralized neur S62597 <1e-100 +24 3097 modifier ofmdg4 mod(mdg4) U30905 <1e-100 intron 3120 string stg X57495 3e-92 −1353127 Histone H2A variant His2AvD X07485 <1e-100 +2 3129 stonewall stwlU41367 <1e-100 −111 3165 tramtrack ttk X71626 1e-15 −416 3205 neuralizedneur X61617 <1e-100 −69 3230 groucho gro M20571 6e-76 intron

[0045] TABLE 3 Insertion locus GS line ESTs Accession Score Insertionsite* 1027 LD12308 AA438512 2e-63 −93 1084 LD12308 AA438512 1e-63 −931135 LD22118 AA817082 <1e-100 −74 2002 LD29847 AA949818 <1e-100 −37 2003LD27171 AA941860 <1e-100 +290 2007 LD06340 AA263242 8e-25 −250 2025LD12957 AA438639 3e-88 +37 2038 LD01639 AA735228 <1e-100 −29 2048ALD03829 AA201147 2e-49 +101 2048B LD07122 AA263935 3e-30 −19 2053LD04728 AA201504 <1e-100 −3 2055 LD03274 AA390332 <1e-100 −51 2067GM02209 AA567240 <1e-100 +56 2074 LD03274 AA390332 <1e-100 −51 2091LD03274 AA390332 <1e-100 −51 2121 LD29214 AA952141 3e-49 +35 2138LD04967 AA201761 6e-53 +362 2163A LD04971 AA201765 <1e-100 −6 2191LD06340 AA263242 6e-82 −275 2202 LD14959 AA440376 <1e-100 intron 2207AGM09451 AA697215 1e-64 −275 2207B LD06340 AA263242 8e-25 −255 2208LD20843 AA541057 4e-62 −11 2209 LD33989 AA979429 <1e-100 +6 3005 LD09360AA390491 <1e-100 −1 3011 LD25593 AA941450 <1e-100 +64 3028 GM10514AA803288 <1e-100 +64 3082 LD14744 AA440145 <1e-100 intron 3086 HL04053AA698259 4e-92 intron 3087 LD32772 AA951892 2e-29 −34 3130 LD02456AA202301 6e-26 within 3199 LD21713 AA735667 <1e-100 −56 3219 LD07107AA263927 <1e-100 within

[0046] By the screening, furthermore, two novel Drosophila genes withsequences analogous to human genes were detected. More specifically, theDrosophila genes were analogous to human Ras associated protein 2 (Ras2)gene (Oncogene 3:201-204, 1988) and human microsome glutathioneS-transferase (mGST) gene (J. Biol. Chem. 263:8430-8436, 1988). Theamino acid sequences of proteins speculated from these Drosophila genesare shown in FIG. 2, compared with the amino acid sequences of theproteins encoded by the human genes.

[0047] Additionally, these novel Drosophila genes were inserted upstreamthe protein-encoding region; after mating with sev-GAL4 line, thesegenes were expressed excessively and detected as rough eye phenotype.

[0048] As has been described above, the invention provides a gene searchmethod comprising P-element insertion and being capable of efficientlyidentifying a novel gene regulating various biological functions ofDrosophila and specifying a function corresponding to the novel gene.

1 6 1 23 DNA Artificial Sequence Description of Artificial SequenceSYNTHESIZED OLIGONUCLEOTIDE 1 ctgaataggg aattgggaat tcg 23 2 45 DNAArtificial Sequence Description of Artificial Sequence SYNTHESIZEDOLIGONUCLEOTIDE 2 aactggaaga attcgcggcc gcaggaattt tttttttttt ttttt 45 322 DNA Artificial Sequence Description of Artificial SequenceSYNTHESIZED OLIGONUCLEOTIDE 3 gtgtatactt cggtaagctt cg 22 4 23 DNAArtificial Sequence Description of Artificial Sequence SYNTHESIZEDOLIGONUCLEOTIDE 4 attgcaagca tacgttaagt gga 23 5 20 DNA ArtificialSequence Description of Artificial Sequence SYNTHESIZED OLIGONUCLEOTIDE5 agaactggaa gaattcgcgg 20 6 21 DNA Artificial Sequence Description ofArtificial Sequence SYNTHESIZED OLIGONUCLEOTIDE 6 cgacgggacc accttatgtta 21

What is claimed is:
 1. A gene search vector, which carries P-elementsequences and two sets of an expression regulatory sequence comprising aUAS sequence for GAL4 transcription activator and a promoter sequence,wherein the expression regulatory sequences are integrated in theP-element sequence in such a manner that their downstreams are inopposite directions.
 2. A gene search vector of claim 1, wherein thepromoter sequence is the core promoter sequence derived from heat shockprotein hsp70 gene.
 3. A gene search vector of claim 1 or 2, wherein thetwo sets of the expression regulatory sequence are independentlyintegrated in the 5′P-element sequence and 3′P-element sequence.
 4. Agene search vector of any one of claims 1 to 3, the gene search vectoradditionally comprising a marker gene between the two sets of theexpression regulatory sequence.
 5. A gene search vector of claim 4,wherein the marker gone is Drosophila white gene.
 6. A gene searchvector of any one of claims 1 to 5, of which the full length is lessthan 8 kbp.
 7. A fly carrying the gene search vector of any one ofclaims 1 to 6 being integrated in the genome thereof.
 8. A method forsearching an unknown gene of Drosophila, comprising the steps of: (a)inserting the gene search vector of any one of claims 1 to 6 into thegenome of Drosophila, (b) mating the vector-inserted Drosophila with aDrosophila expressing the GAL4 transcription activator to createprogenies, and identifying a vector-inserted line with a phenotypedifferent from those of wild-type Drosophila, and (c) determining thenucleotide sequence of the gene for the phenotype of the mutantindividual.
 9. The method according to claim 8, wherein in the step (c),a mRNA fragment including mRNA transcribed from a partial sequence ofthe gene search vector is amplified by reverse-transcriptase-polymerasechain reaction method to determine the nucleotide sequence of theresulting amplified DNA.